cDNA sequence analysis of a 29-kDa cysteine-rich surface antigen of pathogenic Entamoeba histolytica

Journal Article · Wed Aug 01 00:00:00 EDT 1990 · Proceedings of the National Academy of Sciences of the United States of America; (USA)

DOI:https://doi.org/10.1073/pnas.87.16.6358· OSTI ID:5628558

Torian, B E; Stroeher, V L; Stamm, W E ^[1]; Flores, B M ^[2]; Hagen, F S ^[3]

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1. Univ. of Washington, Seattle (USA)
2. Louisiana State Univ. Medical Center, New Orleans (USA)
3. Zymogenetics Incorporated, Seattle, WA (USA)

A {lambda}gt11 cDNA library was constructed from poly(U)-Spharose-selected Entamoeba histolytica trophozoite RNA in order to clone and identify surface antigens. The library was screened with rabbit polyclonal anti-E. histolytica serum. A 700-base-pair cDNA insert was isolated and the nucleotide sequence was determined. The deduced amino acid sequence of the cDNA revealed a cysteine-rich protein. DNA hybridizations showed that the gene was specific to E. histolytica since the cDNA probe reacted with DNA from four axenic strains of E. histolytica but did not react with DNA from Entamoeba invadens, Acanthamoeba castellanii, or Trichomonas vaginalis. The insert was subcloned into the expression vector pGEX-1 and the protein was expressed as a fusion with the C terminus of glutathione S-transferase. Purified fusion protein was used to generate 22 monoclonal antibodies (mAbs) and a mouse polyclonal antiserum specific for the E. histolytica portion of the fusion protein. A 29-kDa protein was identified as a surface antigen when mAbs were used to immunoprecipitate the antigen from metabolically {sup 35}S-labeled live trophozoites. The surface location of the antigen was corroborated by mAb immunoprecipitation of a 29-kDa protein from surface-{sup 125}I-labeled whole trophozoites as well as by the reaction of mAbs with live trophozoites in an indirect immunofluorescence assay performed at 4{degree}C. Immunoblotting with mAbs demonstrated that the antigen was present on four axenic isolates tested. mAbs recognized epitopes on the 29-kDa native antigen on some but not all clinical isolates tested.

• OSTI ID: 5628558
• Journal Information: Proceedings of the National Academy of Sciences of the United States of America; (USA), Vol. 87:16; ISSN 0027-8424
• Country of Publication: United States
• Language: English

Related Subjects

59 BASIC BIOLOGICAL SCIENCES
ANTIGENS
RADIOASSAY
RECOMBINANT DNA
DNA SEQUENCING
AMOEBA
CYSTEINE
DNA HYBRIDIZATION
IMMUNOLOGY
IODINE 125
MONOCLONAL ANTIBODIES
PARASITES
PHOSPHORUS 32
PRECIPITATION
SULFUR 35
AMINO ACIDS
ANIMALS
ANTIBODIES
BETA DECAY RADIOISOTOPES
BETA-MINUS DECAY RADIOISOTOPES
CARBOXYLIC ACIDS
DAYS LIVING RADIOISOTOPES
DNA
ELECTRON CAPTURE RADIOISOTOPES
EVEN-ODD NUCLEI
HYBRIDIZATION
INTERMEDIATE MASS NUCLEI
INVERTEBRATES
IODINE ISOTOPES
ISOTOPES
LIGHT NUCLEI
MICROORGANISMS
NUCLEI
NUCLEIC ACIDS
ODD-EVEN NUCLEI
ODD-ODD NUCLEI
ORGANIC ACIDS
ORGANIC COMPOUNDS
ORGANIC SULFUR COMPOUNDS
PHOSPHORUS ISOTOPES
PROTOZOA
RADIOISOTOPES
SARCODINA
SEPARATION PROCESSES
STRUCTURAL CHEMICAL ANALYSIS
SULFUR ISOTOPES
THIOLS
550201* - Biochemistry- Tracer Techniques